Non-viral vectors for gene-based therapy.

Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions. This modulation is accomplished by introducing exogenous nucleic acids such as DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides. Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors. In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials. The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches.

Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA Induces Rapid and Transient Expression.

Nanoparticle-based delivery of nucleotides offers an alternative to viral vectors for gene therapy. We report highly efficient in vivo delivery of modified mRNA (modRNA) to rat and pig myocardium using formulated lipidoid nanoparticles (FLNP). Direct myocardial injection of FLNP containing 1-10 µg eGFPmodRNA in the rat (n = 3 per group) showed dose-dependent enhanced green fluorescent protein (eGFP) mRNA levels in heart tissue 20 hours after injection, over 60-fold higher than for naked modRNA. Off-target expression, including lung, liver, and spleen, was <10% of that in heart. Expression kinetics after injecting 5 µg FLNP/eGFPmodRNA showed robust expression at 6 hours that reduced by half at 48 hours and was barely detectable at 2 weeks. Intracoronary administration of 10 µg FLNP/eGFPmodRNA also proved successful, although cardiac expression of eGFP mRNA at 20 hours was lower than direct injection, and off-target expression was correspondingly higher. Findings were confirmed in a pilot study in pigs using direct myocardial injection as well as percutaneous intracoronary delivery, in healthy and myocardial infarction models, achieving expression throughout the ventricular wall. Fluorescence microscopy revealed GFP-positive cardiomyocytes in treated hearts. This nanoparticle-enabled approach for highly efficient, rapid and short-term mRNA expression in the heart offers new opportunities to optimize gene therapies for enhancing cardiac function and regeneration.

Dendrimeric siRNA for Efficient Gene Silencing.

Programmable molecular self-assembly of siRNA molecules provides precisely controlled generation of dendrimeric siRNA nanostructures. The second-generation dendrimers of siRNA can be effectively complexed with a low-molecular-weight, cationic polymer (poly(ß-amino ester), PBAE) to generate stable nanostructures about 160 nm in diameter via strong electrostatic interactions. Condensation and gene silencing efficiencies increase with the increased generation of siRNA dendrimers due to a high charge density and structural flexibility.

Lipidoid-coated iron oxide nanoparticles for efficient DNA and siRNA delivery.

The safe, targeted and effective delivery of gene therapeutics remains a significant barrier to their broad clinical application. Here we develop a magnetic nucleic acid delivery system composed of iron oxide nanoparticles and cationic lipid-like materials termed lipidoids. Coated nanoparticles are capable of delivering DNA and siRNA to cells in culture. The mean hydrodynamic size of these nanoparticles was systematically varied and optimized for delivery. While nanoparticles of different sizes showed similar siRNA delivery efficiency, nanoparticles of 50-100 nm displayed optimal DNA delivery activity. The application of an external magnetic field significantly enhanced the efficiency of nucleic acid delivery, with performance exceeding that of the commercially available lipid-based reagent, Lipofectamine 2000. The iron oxide nanoparticle delivery platform developed here offers the potential for magnetically guided targeting, as well as an opportunity to combine gene therapy with MRI imaging and magnetic hyperthermia.

Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation.

The discovery of potent new materials for in vivo delivery of nucleic acids depends upon successful formulation of the active molecules into a dosage form suitable for the physiological environment. Because of the inefficiencies of current formulation methods, materials are usually first evaluated for in vitro delivery efficacy as simple ionic complexes with the nucleic acids (lipoplexes). The predictive value of such assays, however, has never been systematically studied. Here, for the first time, by developing a microfluidic method that allowed the rapid preparation of high-quality siRNA-containing lipid nanoparticles (LNPs) for a large number of materials, we have shown that gene silencing assays employing lipoplexes result in a high rate of false negatives (~90%) that can largely be avoided through formulation. Seven novel materials with in vivo gene silencing potencies of >90% at a dose of 1.0 mg/kg in mice were discovered. This method will facilitate the discovery of next-generation reagents for LNP-mediated nucleic acid delivery.

Lipidoid mRNA Nanoparticles for Myocardial Delivery in Rodents.

An area of active research in the field of cardiac gene therapy aims to achieve high transfection efficiency without eliciting immune or inflammatory reactions. Nanomedicine offers an attractive alternative to traditional viral delivery vehicles because nanoparticle technology can enable safer and more controlled delivery of therapeutic agents. Here we describe the use of lipidoid nanoparticles for delivery of modified mRNA (modRNA) to the myocardium in vivo, with a focus on rodent models that represent a first step toward preclinical studies. Three major procedures are discussed in this chapter: (1) preparation of lipid modRNA nanoparticles, (2) intramyocardial delivery of the lipid modRNA nanoparticles by direct injection with an open chest technique in rats, and (3) intracoronary delivery of the lipid modRNA nanoparticles with open chest and temporary aortic cross clamping in rats.

Stem cell factor gene transfer improves cardiac function after myocardial infarction in swine.

BACKGROUND: Stem cell factor (SCF), a ligand of the c-kit receptor, is a critical cytokine, which contributes to cell migration, proliferation, and survival. It has been shown that SCF expression increases after myocardial infarction (MI) and may be involved in cardiac repair. The aim of this study was to determine whether gene transfer of membrane-bound human SCF improves cardiac function in a large animal model of MI. METHODS AND RESULTS: A transmural MI was created by implanting an embolic coil in the left anterior descending artery in Yorkshire pigs. One week after the MI, the pigs received direct intramyocardial injections of either a recombinant adenovirus encoding for SCF (Ad.SCF, n=9) or ß-gal (Ad.ß-gal, n=6) into the infarct border area. At 3 months post-MI, ejection fraction increased by 12% relative to baseline after Ad.SCF therapy, whereas it decreased by 4.2% (P=0.004) in pigs treated with Ad.ß-gal. Preload-recruitable stroke work was significantly higher in pigs after SCF treatment (Ad.SCF, 55.5±11.6 mm Hg versus Ad.ß-gal, 31.6±12.6 mm Hg, P=0.005), indicating enhanced cardiac function. Histological analyses confirmed the recruitment of c-kit(+) cells as well as a reduced degree of apoptosis 1 week after Ad.SCF injection. In addition, increased capillary density compared with pigs treated with Ad.ß-gal was found at 3 months and suggests an angiogenic role of SCF. CONCLUSIONS: Local overexpression of SCF post-MI induces the recruitment of c-kit(+) cells at the infarct border area acutely. In the chronic stages, SCF gene transfer was associated with improved cardiac function in a preclinical model of ischemic cardiomyopathy.

Niemann-Pick C1 affects the gene delivery efficacy of degradable polymeric nanoparticles.

Despite intensive research effort, the rational design of improved nanoparticulate drug carriers remains challenging, in part due to a limited understanding of the determinants of nanoparticle entry and transport in target cells. Recent studies have shown that Niemann-Pick C1 (NPC1), the lysosome membrane protein that mediates trafficking of cholesterol in cells, is involved in the endosomal escape and subsequent infection caused by filoviruses, and that its absence promotes the retention and efficacy of lipid nanoparticles encapsulating siRNA. Here, we report that NPC1 deficiency results in dramatic reduction in internalization and transfection efficiency mediated by degradable cationic gene delivery polymers, poly(ß-amino ester)s (PBAEs). PBAEs utilized cholesterol and dynamin-dependent endocytosis pathways, and these were found to be heavily compromised in NPC1-deficient cells. In contrast, the absence of NPC1 had minor effects on DNA uptake mediated by polyethylenimine or Lipofectamine 2000. Strikingly, stable overexpression of human NPC1 in chinese hamster ovary cells was associated with enhanced gene uptake (3-fold) and transfection (10-fold) by PBAEs. These findings reveal a role of NPC1 in the regulation of endocytic mechanisms affecting nanoparticle trafficking. We hypothesize that in-depth understanding sites of entry and endosomal escape may lead to highly efficient nanotechnologies for drug delivery.

Nucleic acid-mediated intracellular protein delivery by lipid-like nanoparticles.

Intracellular protein delivery has potential biotechnological and therapeutic application, but remains technically challenging. In contrast, a plethora of nucleic acid carriers have been developed, with lipid-based nanoparticles (LNPs) among the most clinically advanced reagents for oligonucleotide delivery. Here, we validate the hypothesis that oligonucleotides can serve as packaging materials to facilitate protein entrapment within and intracellular delivery by LNPs. Using two distinct model proteins, horseradish peroxidase and NeutrAvidin, we demonstrate that LNPs can yield efficient intracellular protein delivery in vitro when one or more oligonucleotides have been conjugated to the protein cargo. Moreover, in experiments with NeutrAvidin in vivo, we show that oligonucleotide conjugation significantly enhances LNP-mediated protein uptake within various spleen cell populations, suggesting that this approach may be particularly suitable for improved delivery of protein-based vaccines to antigen-presenting cells.

Lipid-like nanomaterials for simultaneous gene expression and silencing in vivo.

New lipid-like nanomaterials are developed to simultaneously regulate expression of multiple genes. Self-assembled nanoparticles are capable of efficiently encapsulating pDNA and siRNA. These nanoparticles are shown to induce simultaneous gene expression and silencing both in vitro and in vivo.

Degradable terpolymers with alkyl side chains demonstrate enhanced gene delivery potency and nanoparticle stability.

Degradable, cationic poly(ß-amino ester)s (PBAEs) with alkyl side chains are developed for non-viral gene delivery. Nanoparticles formed from these PBAE terpolymers exhibit significantly enhanced DNA transfection potency and resistance to aggregation. These hydrophobic PBAE terpolymers, but not PBAEs lacking alkyl side chains, support interaction with PEG-lipid conjugates, facilitating their functionalization with shielding and targeting moieties and accelerating the in vivo translation of these materials.

Effect of molecular weight of amine end-modified poly(ß-amino ester)s on gene delivery efficiency and toxicity.

Amine end-modified poly(ß-amino ester)s (PBAEs) have generated interest as efficient, biodegradable polymeric carriers for plasmid DNA (pDNA). For cationic, non-degradable polymers, such as polyethylenimine (PEI), the polymer molecular weight (MW) and molecular weight distribution (MWD) significantly affect transfection activity and cytotoxicity. The effect of MW on DNA transfection activity for PBAEs has been less well studied. We applied two strategies to obtain amine end-modified PBAEs varying in MW. In one approach, we synthesized four amine end-modified PBAEs with each at 15 different monomer molar ratios, and observed that polymers of intermediate length mediated optimal DNA transfection in HeLa cells. Biophysical characterization of these feed ratio variants suggested that optimal performance was related to higher DNA complexation efficiency and smaller nanoparticle size, but not to nanoparticle charge. In a second approach, we used preparative size exclusion chromatography (SEC) to obtain well-defined, monodisperse polymer fractions. We observed that the transfection activities of size-fractionated PBAEs generally increased with MW, a trend that was weakly associated with an increase in DNA binding efficiency. Furthermore, this approach allowed for the isolation of polymer fractions with greater transfection potency than the starting material. For researchers working with gene delivery polymers synthesized by step-growth polymerization, our data highlight the potentially broad utility of preparative SEC to isolate monodisperse polymers with improved properties. Overall, these results help to elucidate the influence of polymer MWD on nucleic acid delivery and provide insight toward the rational design of next-generation materials for gene therapy.

Alkane-modified short polyethyleneimine for siRNA delivery.

RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer-lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials.

CanScript, an 18-Base pair DNA sequence, boosts tumor cell-specific promoter activity: implications for targeted gene therapy.

Gene therapy protocols for the treatment of cancer often employ gene promoter sequences that are known to be over-expressed in specific tumor cell types relative to normal cells. These promoters, while specific, are often weakly active. It would be desirable to increase the activity of such promoters, while at the same time retain specificity, so that the therapeutic gene is more robustly expressed. Using a luciferase reporter DNA construct in both in vitro cell transfection assays and in vivo mouse tumor models, we have determined that in the absence of any other DNA sequence, a previously identified 18-base pair enhancer sequence called CanScript, lying upstream of the MSLN gene, has ~25% of the promoter activity of CAG, a very strong non-specific promoter/enhancer, in tumor cells in which MSLN is highly expressed. Furthermore, tandem repeat copies of CanScript enhance transcription in a dose-dependent manner and, when coupled with promoter sequences that are active in tumor cells, increase promoter activity. These findings suggest that the incorporation of CanScript into gene constructs may have application in enhancing activity of promoters used in cancer-targeting gene therapy strategies, thereby improving therapeutic efficacy.

Small-Molecule End-Groups of Linear Polymer Determine Cell-type Gene-Delivery Efficacy.

End-modified polymers are promising for the nonviral delivery of genes to cancer cells, immune cells, and human stem cells and point to polymer end-groups as regulators for cell-type specificity. A library of polymers has been synthesized and, although some polymers are strong transfection agents overall, for each cell type, a particular polymer is most effective.